重组蛋白需要同时跨越内膜和外膜两道障碍才能在大肠杆菌中实现胞外分泌。尽管一些重组蛋白由周质空间通过"渗漏"或未知方式实现了跨外膜转运，但机制不明。最近，我们发现两种丝氨酸蛋白酶在大肠杆菌中实现了胞外分泌，并且证明了N端前肽具有介导重组丝氨酸蛋白酶进行跨外膜转运的功能，这一现象和其中蕴含的机制尚无报道。本项目将利用这两种在大肠杆菌中具备不同胞外分泌效率的同源丝氨酸蛋白酶开展研究，在解析它们跨外膜转运的途径和方式的基础上，通过突变和构建嵌合体等手段寻找存在于N端前肽及其他结构区域的与跨外膜转运相关的关键结构特征，进而阐明重组丝氨酸蛋白酶跨外膜转运的分子机制。本项目的开展，有望为人们充分认识重组蛋白通过"渗漏" 或"机制不明"的方式实现胞外分泌这一现象中所蕴含的规律提供新见解。同时，对N端前肽介导重组酶跨外膜转运作用机制深入理解，在应用方面将为以大肠杆菌作为宿主高效分泌重组蛋白提供新的理论依据

Eschericha coli is the most frequently used prokaryotic host for producing recombinant proteins. Extracellular production of recombinant proteins in E. coli has several advantages including rapid preparation and easy purification of the target protein, but remains problematic, mainly due to the presence of the outer membrane. Nevertheless, some recombinant proteins targeting to the periplasmic space of E. coli were found to leak into the culture medium. However, little is known about the mechanism for the "leakage" of recombinant proteins from the periplasmic space to the extracellular milieu. Recently, we found that two bacterial serine proteases, Als and WF146, had the ability to secrete into the culture medium when expressed in E. coli. Meanwhile, we noticed that the secretion efficiency of recombinant Als protease is significanly higher than that of recombinant WF146 protease. Interestingly, the N-terminal propeptide of Als was confirmed to mediate the transport of recombinant Als across the outer membrane of E. coli. It is well known that N-terminal propeptides of bacterial serine proteases usually act as an intramolecular chaperone and an inhibitor of its cognate enzyme, but so far there is no report on N-terminal propeptide-mediated transport of recombinant serine protease. In this research proposal, we are going to study the mechanism of the N-terminal propeptide-mediated transport of recombinant serine protease across the outer membrane of Escherichia coli. Firstly, we will probe possible transport pathway of the enzymes across the outer membrane, and will investigate the role of the N-terminal propeptide in this process. Then, a series of mutants and chimera of the two homologous Als and WF146 proteases will be constructed, in order to indentify the key structural elements involved in transport across the outer membrane. Finally, we will improve the extracellular production level of the recombinant enzymes by optimazing the key structural elements, and by overexpressing transport channel apparatus on the outer membrane. We hope this study could provide new insight into the molecular basis of transport of recombinant serine protease across the outer membrane of E. coli. In addition, the elucidation of the transport mechanism and the roles of the N-terminal propeptide is expected to provide a rational basis for developing a system for high-level extracellular production of recombinant proteins in E. coli.